Method for preparing water-soluble free amine chitosan

ABSTRACT

A method for the preparation of water-soluble chitosan with high purity and biological activity includes steps of: reacting chitosan oligo sugar acid salt, covalently bonded to an organic or inorganic acid, with trialkylamine in phosphate buffered saline followed by addition of an organic solvent to remove acid salt at C-2 position; adding the thus obtained reaction mixture to an inorganic acid to remove trialkylamine salt at C-6 position; and passing the thus obtained free amine chitosan through an activated carbon/ion exchange resin. The free amine chitosan is water-soluble and has a high bioavailability for application to the medicine and food industries.

This patent application claims the benefit of priority from KoreanPatent Application Nos. 2001-59282 filed on Sep. 25, 2001 and 2001-70052filed on Nov. 12, 2001 through PCT Application Serial No. PCTKR02/00694filed on Apr. 16, 2002, the contents of each of which are incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to a method for preparing water-solublefree amine chitosan with high purity.

BACKGROUND OF THE INVENTION

Chitosan is a highly insoluble N-acetylated polymer ofbeta-(1,4)-D-glucosamine with a pyranose unit and a biopolymer with ahigh molecular weight over 1 million Daltons bonded to a functionalgroup of glucosamine over 5,000 units. Chitosan is a cellulose-likepolymer present in fungal walls and the exoskeletons of arthropods,including insects, crabs, shrimps, and lobsters. Chitosan having astructural unit similar to cellulose can be applied in numerous fieldsof medical industry, due to its high bioavailability and a negativeeffect against immune response. Ever since the FDA (of the U.S.)recognized chitosan as a food additive, chitosan has been applied tobiological engineering as an essential biomedical material.

For example, chitosan can be used in various industrial applications,including wastewater treatment (as a coagulant agent, heavy-metalabsorbent, and waste-dye clarifier) and uses in the agriculturalindustry (as a soil-conditioner insecticide, plant antiviral agent, andagricultural chemical agent).

Lately, as the chitosan is known to have a molecular weight of20,000˜100,000 with a highly physical activity, the applications ofchitosan were extended to include the practical fields of foods andbeverages, health and sanitation, cosmetics, textiles, and medicines.The solubility and viscosity of chitosan are considered majorproblematic matters in such applications, since the chitosan iswater-immiscible and grows viscous even when dissolved in water.Therefore, there is an urgent need to provide water-soluble chitosanfree of the above problems.

Water-soluble chitosan has received considerable attention in thiscontext since it can be applied to the aforementioned practical fields.

In the food industry, especially in health food, water-soluble chitosanshows various physical properties, including cholesterol reduction,enhanced immune response, improved liver function, absorption in thealimentary canal, and the control of blood sugar level and bloodpressure. Additionally, chitosan's unique abilities, such as removal ofskin-waste, excellent heat conservation, moisturization, antibacterialactivity, and activation of cutaneous cells, have resulted in itsemployment as a primary component in the cosmetic industry, in suchproducts as shampoo, hair rinse, hair spray, hair gel, creams, bathingagents, face packs, and face-washing agents. Moreover, thewater-solubility of the chitosan is necessary for applications ofproducts administered in a spray form, so that the chitosan can easilypenetrate the wall of inner cutaneous cells and to prevent an increasein viscosity.

Additionally, the water-soluble chitosan is useful as a drug carriersuch as an osteoporosis agent, an antirheumatism treating agent, and aheavy-metal removal agent, which can act in vivo, essentially with ahigh biological activity and water-solubility. As a drug carrier, thewater-soluble chitosan is prepared in a variety of forms includingtablets, capsules, pills, suspensions, solutions, and emulsionsadministered orally and parenterally.

Due to the presence of a strong hydrogen bond among adjacent atoms inchitosan, however, strong acid including organic acids (such as lacticacid, acetic acid, propionic acid, and tartaric acid) and inorganicacids (such as hydrochloric acid, nitric acid, and sulfuric acid) arerequired to dissolve the chitosan. Meanwhile, the toxicity of suchstrong acids raises still another problem to be applied in medicalfields.

There are two different methods to prepare water-soluble chitosan: achemical method and an enzymatic method.

In the chemical method, it is suggested that the water-soluble chitosanis prepared by hydrolysis using hydrochloric acid. This method, however,requires excessive amounts of HCl and an overly long period of time tohydrolyze the chitosan. As an another known chemical method, it is alsosuggested that the water-soluble chitosan is obtained by substitution ofamine moiety at the C-2 position of chitosan with another moiety or byforming amine salt such as —NH₃ ⁺.CH₃CHOHCOO⁻, —NH₃+.CH3COO—, —NH₃⁺.Cl⁻, —NH₃ ⁺.CH₃CH₂COO⁻, —NH₃ ⁺.HCOO⁻, or —NH₃ ⁺. .HOOC(CHOH₂)COO⁻.This process has an advantage in that the water-soluble chitosanprepared does not further reduce molecular weight so that can begenerally used in this art. The water-soluble chitosan obtained by thisprocess, however, has a disadvantage in that it is difficult to dissolvein a gastro-instestinal tract of pH 1.2˜1.5, because the aqueoussolution containing chitosan salt shows pH 3.0 based on a 1.0% solution.

On the contrary, in the enzymatic method, the water-soluble is preparedby enzymatic treatment comprising steps of: dissolving chitosan in pooracid solution; hydrolyzing by enzymatic treatment about for 24 hours;and freeze-drying followed by packaging. Since the drying procedure iscarried out without additional purification, the final product, whichcontains acid, can create problems when administered due to the acid'stoxicity.

Hence, there is a need for method preparing water-soluble chitosan tosolve the aforementioned problems, namely, for effective removal ofleftover impurities and acid salt. As one attempt to realize such amethod, Korea Patent No. 2000-15959 discloses a method for the removalof leftover salt and the unpleasant odor, comprising enzymolysisfollowed by forming an aqueous chitosan oligo sugar solution and passingthe obtained solution through anionic exchange resin. The abovedisclosure, however, neither describes nor suggests properties ofchitosan with high purity.

Therefore, the present invention contrives to realize water-solublechitosan with high biological activity and high purity, one that issuitable for biomedical engineering applications.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method forpreparing water-soluble free amine chitosan.

It is another object of the present invention to provide water-solublefree amine chitosan with a high yield and purity.

It is yet another object of the present invention to providewater-soluble free amine chitosan having a high physiological activity.

Therefore, the present invention provides a method for preparingwater-soluble free amine chitosan, comprising the steps of:

-   -   (a) reacting chitosan oligo sugar acid salt, covalently bonded        to an organic or inorganic acid, with trialkylamine in phosphate        buffered saline, followed by addition an organic solvent to        remove acid salt at the C-2 position;    -   (b) adding the thus obtained reaction mixture to an inorganic        acid to remove trialkylamine salt at the C-6 position; and    -   (c) passing the thus obtained chitosan with free-amine through        an activated carbon/ion exchange resin.

The water-soluble free amine chitosan according to present invention hasfrom 1,000 to 100,000 Daltons of molecular weight in various qualitativeand quantitative analyses.

DETAILED DESCRIPTION OF THE INVENTION

Referring to FIG. 1, the water-soluble free amine chitosan (500) isprepared by: chitosan oligo sugar acid salt (100), as a startingmaterial reacts with trialkylamine followed by adding organic solvent toremove acid salt at C-2 position (200); adding inorganic acid to removetrialkylamine at C-6 position (300); and purification (400).

In step (a) a reaction is carried out by chitosan oligo sugar acid saltreacting with trialkylamine in phosphate buffered saline. Then, thereaction mixture obtained dissolves in a suitable organic solvent,followed by post treatments carried out to remove acid salt.

In a preferred embodiment, as a starting material, the chitosan oligosugar acid salt is prepared by dissolving chitosan in a suitable acid.This is followed by a known method of enzymolysis, whereby the obtainedchitosan solution is treated by a known enzyme. The enzyme is originatedfrom microorganism to convert chitosan oligo sugar and may includechitosanase oriented from Bacillus pumilus, Bacillus subtilis, orBacillus sp. In the above process, chitosan oligo sugar acid salt existsas a complex of both amine at the C-2 position and —CH₂OH at the C-6position with acid salts.

Examples of a suitable acid include organic acid (such as lactic acid,acetic acid, propionic acid, formic acid, ascorbic acid and tartaricacid) and inorganic acid (such as hydrochloric acid, nitric acid, andsulfuric acid). When the lactic acid is used, the amine group at C-2position of the chitosan oligo sugar acid is represented as —NH₃⁺.CH₃CHOHCOO⁻, and the others are as follows.

Organic acid Amine acid salt acetic acid —NH₃ ⁺• CH₃COO⁻ propionic acid—NH₃ ⁺• CH₃CH₂COO⁻ formic acid —NH₃ ⁺• HCOO⁻ ascorbic acid —NH₃ ⁺•CO(COH)₃CHOHCH₂O⁻ tartaric acid —NH₃ ⁺• HOOC(CHOH₂)COO⁻ hydrochloricacid —NH₃ ⁺• Cl⁻ nitric acid —NH₃ ⁺• NO₃ ⁻ sulfuric acid —NH₃ ⁺•HSO₄ ⁻

In step (a), the trialkylamine is used to remove an organic/inorganicacid bonded to amine group at C-2 position and to protect —CH₂OH groupat the C-6 position of chitosan oligo sugar acid salt.

Thus, the trialkylamine reacts with chitosan oligo acid salt, whichexhibits strong acidity, and the amine group among trialkylamineinteracts with H⁺ of the amine group of chitosan oligo sugar acid salt,to be substituted with the alkali group of organic/inorganic acid, i.e.,CH₃CHOHCOO⁻, CH₃COO⁻, and Cl⁻. Therefore, free amine exists at C-2position of chitosan oligo sugar, and at C-6 position, the —CH₂OH group,which is protected by trialkylamine, remains.

Two to three parts (preferably two parts) trialkylamine, relative to onepart of the amine group., is used at C-2 and C-6 positions of chitosanoligo sugar, to effectively remove organic/inorganic acid salt bonded tochitosan oligo sugar. A suitable trialkylamine includes C₁˜C₄ trialkylamine and is preferably trimetylamine, triethylamine, tripropylamine,triisopropylethylamine, or tributylamine; most preferable istriethylamine.

In a preferred embodiment, the reaction in step (a) is carried out inphosphate buffered saline of pH 6.8 to 7.4, preferably 6.8, 7.0, 7.2 or7.4, at room temperature for approximately one to three hours,preferably two hours.

Then, in step (b) to remove acid salt at the C-6 position and anunreacted compound, the reaction mixture is dissolved in a suitableorganic solvent. Examples of a suitable organic solvent include acetone;alcohols such as methanol, ethanol, and propanol; carbon chlorides suchas chloroform and dichloromethane. A known post-treatment method such asair-drying or freeze-drying is subsequently carried out. Here, theair-drying or freeze-drying process is carried out at temperatures of1-30° C. or below −54° C., respectively.

In step (b), an inorganic acid is added to the reaction mixture obtainedin step (a), to remove the trialkylamine salt at the C-6 position andthe unreacted triethylamine.

After trialkylamine salt at C-6 position of chitosan oligo sugar isremoved, chitosan having a structure of free amine at the C-2 positionand a —CH₂OH group at the C-6 position can be obtained.

The employed inorganic acid is hydrochloric acid, nitric acid, orsulfuric acid in the range of 0.0005˜0.6 N. Preferably, hydrochloricacid of 0.0010 N is used.

In a preferred embodiment, the reaction, in step (b), is carried out atroom temperature for approximately one to two hours.

In step (c), the obtained chitosan is purified by passing through anactivated carbon/ion exchange resin. Then, the aforementionedconventional drying process is performed on the resultant. Here, the ionexchange resin may be anionic exchange resin, cationic exchange resin,amphiphilic exchange resin, or non-ionic exchange resin.

Subsequently, a known post-treatment method such as air-drying orfreeze-drying, preferably freeze-drying is subsequently carried out.Here, the air-drying or freeze-drying process is carried out attemperatures of 1-30° C. or below −54° C., respectively.

In other preferred embodiments, the obtained free amine chitosan wasexamined using FT-IR, ¹H-NMR, and ¹³C-NMR techniques and was found to bewater-soluble.

As shown in FIG. 2B, C═O peak (1730 cm⁻¹), originating from an organicacid such as lactic acid, acetic acid, propionic acid, or tartaric acid,disappeared with respect FIG. 2A. At the same time, amide I (C(═O)NH)and amine (—NH₂) peaks, clearly showing water-soluble chitosan, appearat 1635 cm⁻¹ and 1539 cm⁻¹, respectively. In addition, the impurity peakat 2100 cm⁻¹ was remarkably reduced.

In the ¹H-NMR spectrum, it was found that peaks of approximately d1.125-1.360 ppm originated from organic acid and that the impurity peaksdisappeared (see FIGS. 3A and 3B), which is the same tendency as in theabove FT-IR analysis. The ¹H-NMR spectrum analysis also quantitativelyconfirmed the amount of hydrogen present in acetamide.

In the ¹³C-NMR spectrum, it was found that the peak (C═O) originatedfrom organic acid and peaks of impurities were remarkably disappeared(see FIGS. 4A and 4B). The ¹³C-NMR spectrum analysis also quantitativelyconfirmed the amount of hydrogen present in acetamide and amide,respectively. From this spectrometric result, it was found thatwater-soluble free amine chitosan according to the present inventiondoes not contain any organic/inorganic acid and is highly pure.

FIG. 5 shows that the difference of adjacent peak pairs is equal to therepeating unit of glucose amine of chitosan in mass analysis.

As shown in FIGS. 6˜8, it was confirmed that the water-soluble chitosanof the present invention has a molecular weight of 1,000 to 100,000 andexhibits physiological activity.

As aforementioned, the water solubility of the chitosan according to themethod of the present invention can be effectively improved since it hasfree amine with a highly positive charge in chitosan as compared withthat prepared by prior art such as salt formation. In addition, as thechitosan of the present invention has molecular weight with aphysiological activity, it can be preferably applied to medicalapplication. For instance, the chitosan of the present is covalentlybonded to a gene or DNA with a negative charge, such that it can beeasily used as a gene carrier capable of treating diseases based on agene defect or incurable diseases. Further, the application forbiological engineering for use as a biomedical material of thenext-generation is also expected.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which provide a further understanding of theinvention and are incorporated, and constitute a part of thisapplication, illustrate embodiments of the invention and together withthe description serve to explain the principle of the invention. In thedrawings:

FIG. 1 is a flow chart showing a method for preparing water-soluble freeamine chitosan according to the present invention;

FIG. 2A is a graph showing an FT-IR spectrum analysis of chitosan oligosugar acid salt;

FIG. 2B is a graph showing an FT-IR spectrum analysis of free aminechitosan removed acid salt;

FIG. 3A is a graph showing a ¹H-NMR spectrum analysis of chitosan oligosugar with acid salt;

FIG. 3B is a graph showing a ¹H-NMR spectrum analysis of chitosan oligosugar removed acid salt;

FIG. 4A is a graph showing a ¹³C-NMR spectrum analysis of chitosan oligosugar with acid salt;

FIG. 4B is a graph showing a ¹³C-NMR spectrum analysis of free aminechitosan of the invention;

FIG. 5 is a graph showing a mass spectrum analysis of free aminechitosan of the invention: and

FIGS. 6 to 8 are graphs illustrating a molecular weight of free aminechitosan of the invention according to Huggins' equation and Kraemer'sequation.

The invention will be more fully understood by the following examples,which are not to be considered as limiting the scope of the invention.

EXAMPLE 1 Preparation of Water-soluble Free Amine Chitosan

Pre-Step: Preparation of Chitosan Oligo Sugar

Five units of chitosanase originated Bacillus pumilus BN-262 was addedto 100 ml of 5% chitosan solution (pH 5.0˜5.5) dissolved in lactic acidand reacted at 40° C. for 36 hours. After completion of the reaction,the reaction mixture was pre-filtered by 1 μm filter paper andsubsequently filtered using a hollow filter (MW 20,000) [“MW” is theabbreviation for molecular weight]. The thus obtained filtrate wasconcentrated by means of a nano-filter system, sterilized, and then airdried to produce a powdered chitosan oligo sugar protected with lacticacid at the C-2 and C-6 positions.

Step (a): Preparation of Chitosan Oligo Sugar Acid Salt Protected withLactic Acid at C-2 Position

The chitosan oligo sugar in one liter of phosphate buffered saline (pH7.0) was slowly dropped in 0.52 liter of triethylamine. The reaction wascarried out at room temperature for two hours, using two parts oftriethylamine and one part amine of chitosan oligo sugar. Acetone wasadded to the thus obtained reaction mixture while stirring, and theresultant was then centrifuged at 15,000 rpm (using a Supra 30 K) at 4°C. for twenty minutes. After the procedure was repeated three times,air-drying was carried out at room temperature followed by freeze-dryingat a temperature below −54° C., thereby preparing chitosan oligo sugaracid salt having lactic acid at the C-2 position and —CH₂OH group at theC-6 position.

Step (b): Preparation of Free Amine Chitosan at C-2 Position

50 ml of 0.0010 N hydrochloric acid was added to the chitosan oligosugar salt obtained in step (a), and the resultant was reacted for twohours. Acetone was added to the thus obtained reaction mixture whilestirring and then the above-described centrifuging was performed. Afterthe procedure was repeated three times, air-drying was carried out atroom temperature followed by freeze-drying at a temperature below −54°C.

Step (c): Purification

The dried product, prepared as in the step (b), was dissolved indouble-distilled water, which was passed through an ion exchange columnwith activated carbon and then dried, to yield white free aminechitosan.

FT-IR Spectrum Analysis

To compare the chitosan prepared according to the method of the presentinvention with chitosan oligo sugar having organic/inorganic acid, FT-IRanalysis was carried out as follows.

Three milligrams of free amine chitosan and chitosan oligo sugar,prepared as in Example 1, was combined with 300 mg of potassium bromide,ground on an agate mortar and pestle for ten minutes, and molded intopellet. Each pellet was determined by means of FT-IR analysis (using an8700 manufactured by Shimadzu) at 60° C. under reduced pressure.

FIGS. 2A and 2B respectively show the FT-IR spectra of chitosan oligosugar acid salt protected with lactic acid at the C-2 and C-6 positions,prepared per step 1 of Example 1, and free amine chitosan prepared perstep 3 of Example 1.

As shown in FIG. 2A, it was found that the carboxyl group of lactic acidand impurities produced in enzymolysis peak at about 1730 cm⁻¹ and 2100cm⁻¹ (A=0.038), respectively.

On the contrary, as shown in FIG. 2B, the C═O peak (1730 cm⁻¹) of lacticacid almost disappeared and the impurity peaks (2100 cm⁻¹, A=0.024) wereremarkably reduced, as compared with that of FIG. 2A. In addition, dueto hydrogen bond weakened, the amide I (—C═O) and amine (—NH₂) peakappear sharply at about 1635 cm⁻¹ and 1539 cm⁻¹, respectively. Theseresults provide evidence that, according to the method of the presentinvention, the free amine chitosan does not contain any lactic acid oforganic acid, thanks to the complete removal of acid-salt bonded to—CH₂OH and amine at the C-6 position.

¹H-NMR Spectrum Analysis

To compare the free amine chitosan prepared according to the method ofthe present invention with chitosan oligo sugar, ¹H-NMR analysis wascarried out as follows.

Ten milligrams of chitosan oligo sugar (0.54 mmol), prepared per step 1of Example 1, was dissolved in D₂O and then measured by ¹H-NMRspectrometric analyzer (using a DRX-500 manufactured by Bruker). In thesame manner, the free amine chitosan prepared per step 3 of Example 1 isalso detected.

FIGS. 3A and 3B respectively show the ¹H-NMR spectra of chitosan oligosugar with and without acid salt.

In FIG. 3A, it can be seen that lactic acid peaks appear strongly atapproximately d 1.125˜1.360 ppm while the other peaks are impossible todefine due to impurities.

FIG. 3B, however, shows that the lactic acid peaks disappeared and peaksof impurities were remarkably reduced. It was quantitatively confirmedthat one proton at C-1 position (H-1) was appeared at 4.730, 4.684 and4.623 ppm by triplet. Due to oxygen atom with high electro-negativity inglucosamine unit, the resornance of the proton at C-1 position neared byoxygen atom brings out at downfield, therefore, the peak of said protonsplits by triplet at 4.730˜4.623 ppm while the proton of cycloalkaneappears at 0˜2 ppm as usual. Three protons of methyl group bonded tocarbonyl also slightly peaked about at 2.020 ppm by singlet. This peakintensity provides evidence that acetamide group originated from chitinunit of a raw material is quantitatively present. In addition, sixprotons at C-2, C-3, C-4, C-5 and C-6 positions (H-2, H-3, H-4, and H-5)were also appeared about at 3.411˜3.909 ppm. Concretely, due to hydrogenbond weakened, the H-2 peak at C-2 position was divided d 3.910 ppm into3.888 ppm. Also, it was quantitatively found that each H-3, H-4, H-5 andH-6 was split by singlet or doublet.

Further, in FIGS. 3A and 3B, the peak showing at 2.875 ppm appears theprotons of methylene group due to NMR solvent is negligible.

¹³C-NMR Spectrum Analysis

To compare the free amine chitosan according to the present inventionwith chitosan oligo sugar, ¹³C-NMR analysis was carried out as follows.

Ten milligrams of chitosan oligo sugar (0.54 mmol), prepared per step 1of Example 1, was dissolved in D₂O and then measured by means of ¹³C-NMRspectrometric analysis (using a DRX-500 manufactured by Bruker). In thesame manner, the free amine chitosan prepared per step 3 of Example 1 isalso detected.

FIGS. 4A and 4B respectively show the ¹³C-NMR spectra of chitosan oligosugar with and without acid salt.

As shown in FIG. 4A, there is a peak of lactic acid strikingly apparentabout at 20 ppm, while the other peaks are impossible to define due toimpurities.

FIG. 4B, however, quantitatively shows carbon atoms of free aminechitosan prepared by the method of the present invention (per step 3 ofExample 1) and the remaining chitin unit as a raw material. It isquantitatively that peaks of carbon atoms (C-1, C-2, C-3, C-4, C-5 andC-6 positions) of free amine chitosan were appeared about at55.298˜100.699 ppm. Due to oxygen atom with high electro-negativity inglucosamine unit, the resornance of the carbon at C-1 position neared byoxygen atom brings out at downfield, therefore, the peak of said carbonappears at 99.149 ppm while the carbon having sp³ appears at 8˜60 ppm asusual. The impurity peaks were remarkably reduced, as compared with thatof FIG. 4A. Additionally, it is qualitatively found that the peak ofamide I and amide III bonded to carbonyl group, in which originated fromacetamide of chitin unit, appear at 21.520 ppm and 173.995 ppm,respectively.

This result provides evidence that chitin unit in 7% of residue ispresent when 93% degree of deacetylation and 18,579 Daltons of the freeamine chitosan are fully considered.

From these results, it can be seen that water-soluble free aminechitosan according to the present invention does not include anyorganic/inorganic acid and is highly pure.

Mass Analysis

To determine the mass of glucosamine, repeating unit of free aminechitosan according to the present invention, mass spectrometer analysiswas carried out using a mass spectrometer (MALDI-TOF of Shimadzu,Japan).

As shown in FIG. 5, it is found that the molecular weight of free aminechitosan prepared per step 3 in Example 1 is 18,579 Daltons and thedifference between adjacent peak pairs is average 161. The value isequal to that the molecular weight of glucosamine unit of chitosan.

Molecular Weight Analysis

To determine the molecular weight of the free amine chitosan preparedaccording to method of the present invention, specific viscosity inaccordance with concentration was measured using relative viscometer(Vsicotek, Y 501C, USA).

Introducing the value measured Huggins' equation and Kraemer's equationas known in this art, the molecular weight of free amine chitosanprepared per step 3 in Example 1 was confirmed 18,579 Daltons.

EXAMPLE 2 Preparation of Water-soluble Free Amine Chitosan

The water-soluble free amine chitosan was prepared in the same manner asdescribed in Example 1, except that the low molecular weight chitosanoligo sugar as a starting material was used.

To determine the obtained free amine chitosan, the analyses including¹H-NMR, ¹³C-NMR, mass analysis and molecular weight were carried out inthe manner as described in Example 1.

In spectrometric analyses including ¹H NMR and ¹³C NMR, similar patternswere confirmed. In addition, it is found that the free amine chitosan ofExample 2 has 1,246 Daltons (see the FIG. 7).

EXAMPLE 3 Preparation of Water-soluble Free Amine Chitosan

The water-soluble free amine chitosan was prepared in the same manner asdescribed in Example 1, except that the low molecular weight chitosanoligo sugar as a starting material was used.

To determine the obtained free amine chitosan, the analyses including ¹HNMR, ¹³C NMR, mass analysis and molecular weight were carried out in themanner as described in Example 1.

In spectrometric analyses including ¹H NMR and ¹³C NMR, similar patternswere confirmed. In addition, it is found that the free amine chitosan ofExample 3 has 87,753 Daltons (see the FIG. 8).

As described hereinbefore, the water-soluble free amine chitosanaccording to the present invention is prepared by reacting chitosanoligo sugar acid salt with trialkylamine; adding the thus obtainedreaction mixture to inorganic acid to remove acid salt at the C-2position; and passing the thus obtained chitosan through activatedcarbon/ion exchange resin. Also, the water-soluble free amine chitosanaccording to present invention was found to have a molecular weight of1,000˜100,000 Daltons and to exhibit biological activity and highpurity. Exerting a positive charge of free amine of chitosan, the freeamine chitosan according to the present invention can therefore be usedas a gene carrier for DNA with a negative charge, for treatment ofdisease due to gene defects in the medical industry, and as a primarycomponent in the functional food industry.

The forgoing embodiments are merely exemplary and are not to beconstrued as limiting the scope of the present invention. The presentteachings can be readily applied to other types of methods. Thedescription of the present invention is intended to be illustrative, andnot to limit the scope of the claims. Many alternatives, modifications,and variations will be apparent to those skilled in the art.

1. A method for preparing water-soluble chitosan having free aminegroups, comprising the steps of: (a) enzymatically hydrolyzing anorganic or inorganic acid salt of chitosan oligo sugar; (b) reacting thehydrolysis product of step (a) with trialkylamine; (c) adding an organicsolvent to the product of step (b) to remove the acid salt at C-2position of the chitosan oligo sugar; (d) adding an inorganic acid tothe product of step (c) to remove trialkylamine salt at C-6 position ofthe chitosan oligo sugar; and (e) purifying a chitosan having free aminegroups from the products of step (d).
 2. The method according to claim1, wherein the molecular weight of the water-soluble chitosan rangesfrom 1,000 to 100,000 Da.
 3. The method according to claim 1, whereinthe organic acid of said step (a) is selected from the group consistingof lactic acid, acetic acid, propionic acid, formic acid, ascorbic acidand tartaric acid.
 4. The method according to claim 1, wherein theinorganic acid of said step (a) is selected from the group consisting ofhydrochloric acid, nitric acid, and sulfuric acid.
 5. The methodaccording to claim 1, wherein the trialkylamine is selected from thegroup consisting of trimethylamine, triethylamine, tripropylamine,triisopropylethylamine, and tributylamine.
 6. The method according toclaim 1, wherein the trialkylamine is trimethylamine.
 7. The methodaccording to claim 1, wherein the trialkylamine is used in an amount oftwo to three parts relative to one part of the amine group of chitosanoligo sugar acid salt.
 8. The method according to claim 1, wherein saidstep (b) occurs in phosphate buffered saline solution in a range of frompH 6.8 to pH 7.4.
 9. The method according to claim 1, wherein theorganic solvent is selected from the group consisting of acetone;alcohols selected from the group consisting of methanol, ethanol, andpropanol; and carbon chlorides selected from the group consisting ofchloroform and dichloromethane.
 10. The method according to claim 1,wherein the inorganic acid of said step (d) is selected from the groupconsisting of hydrochloric acid, nitric acid, and sulfuric acid.
 11. Themethod according to claim 1, wherein the inorganic acid of said step (d)is hydrochloric acid.
 12. The method according to claim 1, wherein theconcentration of the inorganic acid of said step (d) ranges from 0.0005to 0.600 N.
 13. The method according to claim 1, wherein the organic orinorganic acid salt of chitosan oligo sugar is prepared by dissolvingchitosan oligo sugar in an organic acid or inorganic acid.
 14. Themethod according to claim 1, wherein the organic or inorganic acid saltof chitosan oligo sugar is treated with chitosanase.
 15. The methodaccording to claim 1, wherein the organic or inorganic acid salt ofchitosan oligo sugar is dissolved in phosphate buffered saline solution.16. The method according to claim 1, wherein step (e) includes passingthe product of step (d) through an activated carbon/ion exchange torecover a chitosan having free amine groups.